CN102821597B - Vip3Ab and CRY1Fa is for managing the combined use of resistant insects - Google Patents

Abstract

The present invention includes that the method for controlling lepidopteran insects and plant, described plant comprise the Vip3Ab insecticidal proteins with the combination of Cry1Fa insecticidal proteins to postpone or the formation of prevention insect-resistant.

Description

Combination use of Vip3Ab and CRY1Fa for management of resistant insects

Background of the invention

Humans grow maize for food and energy applications. Humans also grow many other crops, including soybeans and cotton. Insects eat and damage plants, and thereby undercut these human efforts. Billions of dollars are spent each year to control insect pests, and additional billions are lost in the damage they cause. Synthetic organic chemical insecticides have been the main tool for controlling insect pests, but biological insecticides, such as insecticidal proteins derived from Bacillus thuringiensis (Bt), have been used in some fields Play an important role. The ability to generate insect resistant plants via genetic transformation with Bt insecticidal proteins has revolutionized modern agriculture and has increased the importance and value of insecticidal proteins and their genes.

Several Bt proteins have been used to create insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include Cry1Ab, Cry1Ac, Cry1F and Cry3Bb in maize, Cry1Ac and Cry2Ab in cotton, and Cry3A in potato.

Except where a combined insecticidal spectrum of the 2 proteins is desired (for example, Cry1Ab and Cry3Bb in maize combine to provide resistance to lepidopteran pests and rootworms, respectively) or independent actions of the proteins make them useful Commercial products expressing these proteins express a single protein, except in the case of tools to delay resistance development in susceptible insect populations (eg, the combination of Cry1Ac and Cry2Ab in cotton to provide resistance management in tobacco aphids). See also US 20090313717, which relates to the Cry2 protein and Vip3Aa, Cry1F, or Cry1A to control Helicoverpa zea or armigerain. WO 2009 132850 relates to Cry1F or Cry1A and Vip3Aa for the control of Spodoptera frugiperda. US 2008 0311096 relates in part to Cry1Ab to control Cry1F resistant ECB.

That said, some of the quality of insect-resistant transgenic plants that has led to the rapid and widespread adoption of this technology also raises concerns that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies to preserve the utility of Bt-based insect resistance traits have been suggested, including combining with refuges and alternating with different toxins, or deploying high doses of the protein in co-deployment of different toxins (McGaughey et al. et al. (1998), "B.t. Resistance Management," Nature Biotechnol. 16:144-146).

Proteins selected for use in an IRM stack need to exert their insecticidal effects independently such that resistance developed to one protein does not confer resistance to the second protein (i.e., no cross-resistance to proteins) . If, for example, a selected pest population is sensitive to "Protein B" in terms of resistance to "Protein A", then it would be inferred that there is no cross-resistance, and the combination of Protein A and Protein B will effectively delay resistance to Protein A alone. sex.

In the absence of resistant insect populations, assessments can be made based on other characteristics hypothesized to be relevant to the mechanism of action and potential for cross-resistance. The utility of receptor-mediated binding in identifying pesticidal proteins that are likely not to exhibit cross-resistance has been suggested (van Mellaert et al. 1999). A key predictor of the lack of cross-resistance inherent in this approach is that pesticidal proteins do not compete for receptors in sensitive insect species.

Where two Bt toxins compete for the same receptor, then if said receptor is mutated in said insect such that one of the toxins no longer binds to said receptor, and thus is no longer insecticidal against the insect, It may then be the case that the insect will also be resistant to a second toxin that competes for binding to the same receptor. That said, insects are said to be cross-resistant to both Bt toxins. However, if two toxins bind two different receptors, this may be an indication that the insect will not be resistant to both toxins at the same time.

Cry1Fa can be used to control many lepidopteran pest species, including European cornborer (ECB; Ostrinia nubilalis (Hübner)) and fall armyworm (FAW; Spodoptera frugiperda), and targets Sugarcane borer (SCB; Diatraea saccharalis) was active. The CrylFa protein (as produced in maize plants containing event TC1507) is responsible for an industry-leading insect resistance trait for FAW control. Further deployment in SmartStax ^TM , and WideStrike ^TM products.

Because the most common techniques available for labeling proteins detected in receptor binding assays inactivate the insecticidal activity of CrylFa proteins, the ability to use CrylFa proteins for (competitive or homologous) receptor binding studies is limited.

Other Cry toxins are listed on the official B.t. Nomenclature Commission site (Crickmore et al; lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). There are currently almost 60 major classes of "Cry" toxins (Cry1-Cry59), as well as other Cyt toxins and VIP toxins. Many groups of digits each have a subgroup of uppercase letters, and a subgroup of uppercase letters has a subgroup of lowercase letters. (eg Cry1 has A-L and Cry1A has a-i).

Summary of the invention

The present invention relates in part to the surprising discovery that populations of Fall Armyworm (Spodoptera frugiperda; FAW) resistant to the insecticidal activity of the CrylFa protein are not resistant to the insecticidal activity of the Vip3Ab protein. The subject toxin pairs provide non-cross-resistant effects against FAW.

Plants expressing Vip3Ab and Cry1Fa, or pesticidal portions thereof, will be useful to delay or prevent the development of resistance to any of these pesticidal proteins alone, as will be recognized by those skilled in the art with the benefit of this disclosure.

The present invention is also supported by the finding that Vip3Ab and CrylFa do not compete with each other for binding to binding sites in the intestine from FAW.

Thus, the present invention relates in part to the use of the Vip3Ab protein in combination with the CrylFa protein. Plants (and areas of land planted with such plants) that produce Vip3Ab and CrylFa are included within the scope of the present invention.

The present invention also relates in part to triple stacks or "pyramids" of three toxins (or more), wherein Vip3Ab and CrylFa are the base pair. In some preferred pyramid embodiments, selected toxins have non-cross-resistant effects against FAW. Some preferred proteins for these triple stack plus pyramid combinations are Cry1Fa and Vip3Ab and Cry1C, Cry1D, Cry1Be, or Cry1E. According to the present invention, these specific triple stacks advantageously and surprisingly provide a non-cross-resistant effect against FAW. This can help reduce or eliminate the need for shelter land area.

In the case of CrylFa active against both FAW and European corn borer (ECB), and based on the data presented herein, a quadruple (four-way) stack could also be chosen to provide 4 proteins, of which 4 Three of the species had non-cross-resistant activity against ECB, and three of the four species had non-cross-resistant activity against FAW. This can be achieved by using CrylBe (active against both ECB and FAW) and the subject protein pair, plus an additional protein active against ECB. According to the present invention, such quadruple stacks are:

Cry1F plus Cry1Be plus Vip3Ab (active against FAW) plus Cry1Ab, Cry2A, Cry1I or DIG-3 (active against ECB).

Brief description of the drawings

Figure 1: Dose response of full-length Vip3Ab1 against growth inhibition (rods) and mortality (♦) of wild-type Spodoptera frugiperda (J.E.Smith), (FAW) and Cry1Fa-resistant Spodoptera frugiperda (J.E.Smith), (rFAW) . Percent growth inhibition is based on comparing the average weight of 8 larvae treated with buffer only to the weight of larvae exposed to toxin for 5 days.

Figure 2: Phosphorescence image ^of125ICry1Fa bound to BBMV from Spodoptera frugiperda after separation by SDS-PAGE. Samples were done in duplicate. The concentration of ¹²⁵ I CrylFa was 1 nM. Control represents the level ^of125ICrylFa binding to BBMV in the absence of any competing ligand. 1,000 nM CrylFa represents the level of ¹²⁵ I CrylFa binding to BBMV in the presence of 1,000 nM non-radiolabeled CrylFa, showing complete displacement of radiolabeled ligand from BBMV protein. 1,000 nM MVip3Ab1 represents the level of ¹²⁵ I CrylFa binding to BBMV in the presence of 1,000 nM non-radioactively labeled Vip3Ab1, showing that this protein does not displace ¹²⁵ I from Spodoptera frugiperda BBMV even when added at 1,000-fold the concentration of the labeled ligand The ability of Cry1Fa.

Figure 3: Phosphor ^of125I CrylFa bound to BBMV from wild-type Spodoptera frugiperda (FAW) or CrylFa-resistant Spodoptera frugiperda (rFAW) after separation by SDS-PAGE. Samples were done in duplicate. The concentration of ¹²⁵ I CrylFa was 2.5 nM. FAW-0 represents the level at which ¹²⁵ I CrylFa binds wild-type Spodoptera frugiperda BBMV in the absence of any competing ligand. FAW-1,000 nM Cry1Fa represents the level of ¹²⁵ I Cry1Fa binding to wild-type Spodoptera frugiperda BBMV in the presence of 1,000 nM non-radiolabeled Cry1Fa, showing displacement of radiolabeled ligand from BBMV protein. rFAW-0 represents the level of ¹²⁵ I Cry1Fa binding to Cry1Fa-resistant Spodoptera frugiperda BBMV in the absence of any competing ligand. Note the lack ^of125ICry1Fa binding to BBMV from resistant FAW. rFAW-1,000 nM Cry1Fa represents the level at which ¹²⁵ I Cry1Fa binds BBMV in the presence of 1,000 nM non-radiolabeled Vip3Ab1, again showing that ¹²⁵ I Cry1Fa cannot bind BBMV from Cry1Fa resistant Spodoptera frugiperda.

Detailed description of the invention

As reported herein, the Vip3Ab toxin produced in transgenic maize (and other plants; such as cotton and soybean) can be very effective in controlling Fall Armyworm (FAW; Spodoptera frugiperda) that has developed resistance to CrylFa activity . Thus, the present invention relates in part to the surprising discovery that Cry1Fa-resistant Fall Armyworm is susceptible (ie, not cross-resistant) to Vip3Ab. In other words, the present invention also relates in part to the surprising discovery that Vip3Ab toxins effectively protect plants, such as maize plants, from damage by CrylFa resistant Fall Armyworm. For a discussion of this pest, see eg Tabashnik, PNAS (2008), Vol. 105 No. 49, 19029-19030.

The present invention includes the protection of Vip3Ab toxins in maize and other economically important plant species such as soybeans from damage and yield loss caused by Fall Armyworm feeding or against Fall Armyworm populations that have developed resistance to CrylFa use.

Thus, the present invention teaches the stacking of IRMs comprising Vip3Ab to prevent or reduce the development of resistance to Cry1Fa in Fall Armyworm.

The present invention provides a composition for controlling Lepidopteran pests comprising cells that produce a protein comprising a Cry1Fa core toxin and a protein comprising a Vip3Ab core toxin.

The present invention further comprises hosts transformed to produce both the CrylFa pesticidal protein and the Vip3Ab pesticidal protein, wherein said host is a microorganism or a plant cell. Preferably, the subject polynucleotide is under the control of (operably linked to/comprising) a non-Bacillus thuringiensis promoter in the genetic construct. A subject polynucleotide may comprise codon usage for enhanced expression in plants.

In addition, the present invention is intended to provide a method of controlling Lepidoptera pests, comprising contacting said pests or the environment of said pests with an effective amount of a composition comprising a Cry1Fa core toxin-containing protein, and further comprising Protein containing Vip3Ab core toxin.

One embodiment of the invention includes maize plants comprising a plant expressible gene encoding a protein comprising a Vip3Ab core toxin and a plant expressible gene encoding a protein comprising a Cry1Fa core toxin, and seeds of such plants.

Yet another embodiment of the present invention includes maize plants, and seeds of such plants, into which maize have been introgressed with a plant expressible gene encoding a protein comprising the Vip3Ab core toxin and a plant expressible gene encoding a protein comprising the Cry1Fa core toxin in plants.

As described in the Examples, competition binding studies using radiolabeled Vip3Ab core toxin protein showed that the CrylFa core toxin protein did not compete for binding of Vip3Ab binding in FAW insect tissues. These results also indicate that the combination of Cry1Fa and Vip3Ab proteins is an effective means of alleviating the development of resistance to Cry1Fa (and likewise, resistance development to Vip3Ab) in the FAW population, and that it may be possible to increase the expression of these two proteins level of resistance to this pest in maize plants.

Thus, based in part on the data described herein, it was thought that the co-production (stack) of Vip3Ab and CrylFa proteins could be used to generate a high dose IRM stack against FAW. Where CrylFa is active against both FAW and European corn borer (ECB), the subject toxin pair provides a non-competitive effect against FAW.

Additional proteins can be added to this pair to expand the spectrum of insect control.

Another deployment option would be to use the Cry1Fa and Vip3Ab proteins in combination with another third toxin/gene and use this triple stack to mitigate the development of resistance to either of these toxins in FAW. Thus, another deployment option for the present invention would be to use two, three, or more proteins in crop growing areas where FAW can form resistant populations.

Thus, the present invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, wherein the Cry1Fa and Vip3Ab toxins are the base pair.

In some preferred pyramid embodiments, three selected proteins provide non-cross-resistant effects against FAW. Some preferred "triple action" pyramid combinations are Cry1Fa and Vip3Ab and Cry1C or Cry1D. See USSN 61/284,281 (filed December 16, 2009) (which shows that Cry1C is active against Cry1F-resistant FAW) and USSN 61/284,252 (filed December 16, 2009), which shows that Cry1D is active against Cry1F-resistant FAW Sexual FAW is active. These two applications also show that Cry1C does not compete with Cry1F for binding in FAW membrane preparations, and that Cv1D does not compete with Cry1F for binding in FAW membrane preparations. In some embodiments, Cry1Be or Cry1E can be combined with Vip3A and Cry1F (as a third anti-FAW protein). For the use of Cry1Be and Cry1F, see USSN 61/284,290 (filed December 16, 2009). For the use of Cry1E and Cry1F, see USSN 61/284,278 (filed December 16, 2009). According to the present invention, these specific triple stacks advantageously and surprisingly provide three proteins that provide non-cross-resistant effects against FAW. This can help reduce or eliminate the need for shelter land area.

Based on the data presented here, a quadruple (four-position) stack could also be chosen to provide three proteins with non-cross-resistant effects against ECB and three proteins with non-cross-resistant effects against FAW. This can be obtained by using Cry1Be (active against both ECB and FAW) and Cry1Fa (active against both ECB and FAW) and the subject Vip3Ab (active against FAW) and a fourth protein with ECB toxicity (see 2009 USSN 61/284,290, filed December 16, which concerns the combination of Cry1Fa and Cry1Be). An example of a quadruple stack according to the present invention is:

Cry1F plus Cry1Be plus Vip3 (active against FAW) plus (Cry1Ab, Cry2A, Cry1I, or DIG-3 - all active against ECB).

DIG-3 is disclosed in US 2010 00269223.

Plants (and areas of land planted with such plants) that produce any of the subject combinations of proteins are included within the scope of this invention. Other toxins/genes can also be added, but the particular stack discussed above advantageously and surprisingly provides multiple modes of action against FAW and/or ECB. This can help reduce or eliminate the need for shelter land area. Thus, fields greater than 10 acres so planted are included in this invention.

GENBANK can also be used to obtain the sequences of any genes and proteins disclosed or referred to herein. See Appendix A below.

US Patent No. 5,188,960 and US Patent No. 5,827,514 describe CrylFa core toxin-containing proteins suitable for use in the practice of the present invention. US Patent No. 6,218,188 describes plant-optimized DNA sequences encoding CrylFa core toxin-containing proteins suitable for use in the present invention.

Cry1Fa in SmartStax ^TM , and WidesStrike ^TM products. The vip3Ab gene can be combined into e.g. Cry1Fa products such as middle. Thus, the use of Vip3Ab can significantly reduce the selection pressure on these and other commercially available proteins. Thus, Vip3Ab can be used in maize and other plants (eg, cotton and soybean) in 3-gene combinations.

The protein combinations described herein can be used to control Lepidoptera pests. Adult Lepidoptera such as butterflies and moths feed primarily on nectar and are important effectors for pollination. Almost all Lepidopteran larvae, ie caterpillars, feed on plants and many are serious pests. Caterpillars feed on or inside leaves or on plant roots or stems, depriving plants of nutrients and often damaging the plants' physical support structures. In addition, caterpillars feed on fruit, fabric, and stored grain and flour, destroying or severely reducing the value of these products for sale. As used herein, references to Lepidoptera pests refer to the various life stages of the pests, including the larval stage.

Some chimeric toxins of the invention include the entire N-terminal core toxin portion of the Bt toxin, and at some point beyond the end of the core toxin portion, the protein has a transition to a heterologous protoxin sequence. The N-terminal pesticidally active toxin portion of the Bt toxin is referred to as the "core" toxin. The transition from the core toxin segment to the heterologous protoxin segment can occur approximately at the toxin/protoxin junction or, in the alternative, a portion of the native protoxin (extending beyond the core toxin portion) can be retained, where it occurs downstream Transition to the heterologous protoxin moiety.

For example, one chimeric toxin of the invention is the complete core toxin portion of CrylFa (approximately the first 600 amino acids) and a heterologous protoxin (the rest of the C-terminus of the protein). In a preferred embodiment, the portion of the chimeric toxin comprising the protoxin is derived from the Cry1Ab protein toxin. In a preferred embodiment, the portion of the chimeric toxin comprising the protoxin is derived from the Cry1Ab protein toxin.

Those skilled in the art will appreciate that Bt toxins (even within a class such as CrylF) will vary to some extent in the length and precise location of the transition from the core toxin portion to the protoxin portion. Typically, CrylFa toxins are about 1150 to about 1200 amino acids in length. The transition from the core toxin portion to the protoxin portion typically occurs at about 50% to about 60% of the full-length toxin. Chimeric toxins of the invention will include a full expansion of this N-terminal core toxin portion. Thus, the chimeric toxin will comprise at least about 50% of the full length of the CrylFaBt toxin protein. Typically this will be at least about 590 amino acids. Regarding the protoxin portion, the entire Cry1Ab protoxin portion extends from the end of the core toxin portion to the C-terminus of the molecule.

Genes and toxins. Genes and toxins useful in accordance with the present invention include not only the disclosed full-length sequences, but also fragments of these sequences, variants, mutants, and fusion proteins that retain the characteristic pesticidal activity of the toxins specifically exemplified herein . As used herein, the term "variant" or "variation" of a gene refers to a nucleotide sequence encoding the same toxin or an equivalent toxin having pesticidal activity. As used herein, the term "equivalent toxin" refers to a toxin having the same or substantially the same biological activity against a target pest as the claimed toxin.

As used herein, according to "Revision of the Nomenclature for the Bacillusthuringiensis Pesticidal Crystal Proteins," N. Crickmore, D.R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D.H. Dean. Microbiology and Molecular Biology Reviews (1998) Vol. 62:807-813, borders represent approximately 95% (Cry1Fa and Vip3Ab), 78% (Cry1F and Vip3Ab), and 45% (Cry1 and Vip3Ab) sequence identities. These cut-offs may also apply only to core toxins (eg for Vip3Ab).

It should be apparent to those skilled in the art that genes encoding active toxins can be identified and obtained via several means. The specific genes or gene parts exemplified herein can be obtained from isolates deposited in culture depositories. These genes, or parts or variants thereof, can also be constructed synthetically, for example by using a gene synthesizer. Variations of genes can be readily constructed using standard techniques for generating point mutations. Also, fragments of these genes can be generated using commercially available exonucleases or endonucleases according to standard protocols. For example, nucleotides can be systematically truncated from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. Genes encoding active fragments can also be obtained using various restriction enzymes. Active fragments of these protein toxins can be directly obtained using proteases.

Fragments and equivalents that retain the pesticidal activity of the exemplified toxins are intended to be within the scope of the invention. Also, due to the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of the artisan to create these alternative DNA sequences encoding the same, or substantially the same toxin. These variant DNA sequences are within the scope of the present invention. As used herein, reference to "substantially identical" sequences refers to sequences having amino acid substitutions, deletions, additions, or insertions that do not substantially affect pesticidal activity. Fragments of genes encoding proteins that retain pesticidal activity are also included in this definition.

Yet another method for identifying toxin-encoding genes and gene portions useful according to the invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by means of suitable labels or may be generated with intrinsic fluorescence, as described in International Application No. WO93/16094. As is well known in the art, if a probe molecule and a nucleic acid sample hybridize by forming a strong bond between the two molecules, it is reasonable to assume that the probe and sample have substantial homology. Preferably, hybridization is performed under stringent conditions by techniques well known in the art, as described, for example, in Keller, G.H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. Some examples of salt concentration and temperature combinations are as follows (in order of increasing stringency): 2X SSPE or SSC at room temperature; 1X SSPE or SSC at 42°C; 0.1X SSPE or SSC at 42°C; 0.1X SSPE or SSC at 65°C. Detection of the probes provides a means for determining in a known manner whether hybridization has occurred. Such probe analysis provides a rapid method for identifying toxin-encoding genes of the invention. Nucleotide segments for use as probes according to the present invention can be synthesized using a DNA synthesizer and standard protocols. These nucleotide sequences can also be used as PCR primers to amplify the genes of the present invention.

Variant toxin . Certain toxins of the invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the invention, it should be readily apparent that the invention includes variants or equivalent toxins (and nucleotide sequences encoding equivalent toxins) having the same or similar pesticidal activity of the exemplified toxins. . Equivalent toxins will have amino acid homology to the exemplified toxins. Typically this amino acid homology will be greater than 75%, preferably greater than 90%, and most preferably greater than 95%. Amino acid homology will be highest in critical regions of the toxin that are either responsible for biological activity or involved in determining the three-dimensional configuration ultimately responsible for biological activity. Certain amino acid substitutions are acceptable in this regard and may be contemplated if such substitutions are in regions that are not critical for activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids can be placed into the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions in which an amino acid of one class is replaced by another amino acid of the same type fall within the scope of the invention as long as the substitution does not substantially alter the biological activity of the compound. Below is a list of examples of amino acids belonging to each class.

Table 1

Types of Amino Acids Examples of Amino Acids non-polar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp uncharged polarity Gly, Ser, Thr, Cys, Tyr, Asn, Gln acidic Asp,Glu alkaline Lys, Arg, His

In some cases, non-conservative substitutions may also be made. A critical factor is that these substitutions must not significantly reduce the biological activity of the toxin.

recombinant host . Genes encoding the toxins of the invention can be introduced into a wide variety of microbial or plant hosts. Expression of toxin genes leads directly or indirectly to the intracellular production and maintenance of pesticides. Bt strains expressing the two toxins of the invention can be created using conjugative transfer and recombinant transfer. Other host organisms can also be transformed with one or both toxin genes, which are then used to achieve a synergistic effect. With a suitable microbial host, such as Pseudomonas, the microbes can be applied to the pest's locus, where they will proliferate and be ingested. The result is pest control. Alternatively, microorganisms that host toxin genes can be treated under conditions that prolong the activity of the toxin and stabilize the cells. The treated cells (which retain toxic activity) can then be applied to the environment of the target pest.

In the case of introducing a Bt toxin gene into a microbial host via an appropriate vector, and applying the host to a living environment, it is necessary to use some host microorganism. A microbial host is selected that is known to occupy the "phylloplane" (phylloplane, phyllosphere, rhizosphere, and/or root surface) of one or more crops of interest. These microorganisms were selected to enable successful competition with wild-type microorganisms in specific environments (crop and other insect habitats), to provide stable maintenance and expression of genes expressing polypeptide pesticides, and, desirably, to provide improved protection against pesticides. degradation and inactivation in the environment.

A large number of microorganisms are known to reside in the foliage (surface of plant leaves) and/or rhizosphere (soil around plant roots) of a very wide variety of important crops. These microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms such as bacteria, e.g. Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas ( Xanthomonas), Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, especially yeast, such as the genus Saccharomyces ), Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are phytosphere bacterial species such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroids, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Vigne Azotobacter vinlandii; and phytospheric fungal species such as Rhodotorula rubra, R. glutinis, R. marina, Rhodotorula rubra (R.aurantiaca), Cyptococcus albidus, C.diffluens, C.laurentii, Saccharomyces rosei, S.pretoriensis, Saccharomyces cerevisiae (S cerevisiae), Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are pigmented microorganisms.

A wide variety of methods are available for introducing toxin-encoding Bt genes into microbial hosts under conditions that permit stable maintenance and expression of the genes. These methods are well known to those skilled in the art and are described, for example, in US Patent No. 5,135,867, which is incorporated herein by reference.

Cell handling . Bacillus thuringiensis or recombinant cells expressing Bt toxins can be treated to prolong toxin activity and stabilize the cells. The resulting pesticide microcapsules contain the one or more Bt toxins within an already stabilized cellular structure and protect the toxins when the microcapsules are applied to the environment of the target pest. Suitable host cells may include prokaryotes or eukaryotes, and are generally not limited to those cells that do not produce substances that are toxic to higher organisms, such as mammals. However, organisms that produce substances toxic to higher organisms may be used, where the toxic substance is either unstable or applied at sufficiently reduced levels to avoid any possibility of toxicity to mammalian hosts. Of particular interest as hosts will be prokaryotes and lower eukaryotes, such as fungi.

Cells will usually be whole and, upon treatment, in a substantially proliferating form rather than in spore form, although spores may be employed in some cases.

Microbial cells, such as microorganisms containing one or more B.t. Protects against toxic cellular properties. Examples of chemical agents are halogenating agents, especially halogens having an atomic number of 17-80. More particularly, iodine may be used under mild conditions and for a sufficient time such that the desired result is achieved. Other suitable techniques include the use of aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various tissue fixations agents, such as Lugol's iodine, Bouin's fixative, various acids and Helly's fixatives (see: Humason, Gretchen L., Animal Tissue Techniques, W.H. Freeman and Company, 1967); or retain and prolong when cells are applied to the host environment Combined physical (thermal) and chemical agent treatment of the activity of toxins produced in cells. Examples of physical means are short-wavelength radiation, such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treating microbial cells are disclosed in US Patent Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.

Cells generally have increased structural stability, which increases resistance to environmental conditions. Where the pesticide is a proform, the method of cell treatment should be chosen so as not to inhibit processing of the proform by the target pest pathogen into the mature form of the pesticide. Formaldehyde, for example, cross-links proteins and can inhibit the processing of prototype peptide pesticides. Treatment methods should preserve at least a substantial portion of the bioavailability or biological activity of the toxin.

Features of particular interest in selecting host cells for production purposes include ease of introduction of one or more B.t. The presence. Features of interest for use as pesticide microcapsules include pesticide protective qualities, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness of feeding pests; ease of killing and immobilization without damage to toxins; etc. Other considerations include ease of formulation and handling, economy, storage stability, and the like.

cell growth . Cell hosts containing one or more Bt pesticidal genes can be cultured in any convenient nutrient medium in which the DNA construct confers a selective advantage, providing a selective medium such that substantially all or all cells retain the Bt gene. These cells can then be harvested in a conventional manner. Alternatively, cells can be treated prior to harvesting.

B.t. cells producing toxins of the invention can be cultured using standard technical media and fermentation techniques. After completion of the fermentation cycle, bacteria can be harvested by first isolating B.t. spores and crystals from the fermentation medium by means well known in the art. Recovered B.t. spores and crystals can be formulated into wettable powders, liquid concentrates, granules or other formulations by adding surfactants, dispersants, inert carriers and other components to facilitate treatment and treatment of specific target pests. application. Such formulations and application procedures are well known in the art.

formulation . Formulated bait granules containing attractants and Bt isolates, or spores, crystals, and toxins of recombinant microorganisms comprising genes obtainable from the Bt isolates disclosed herein, can be applied to soil. The formulated product can also be applied at a later stage of the crop cycle as a seed coating material or as a root treatment or as a general plant treatment. Plant and soil treatments of Bt cells can be employed as wettable powders, granules or dusts by mixing various inert materials such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, etc. ) or plant material (powdered corn cobs, rice husks, walnut shells, etc.). Formulations may contain spreader-sticker adjuvants, stabilizers, other insecticidal additives, or surfactants. Liquid formulations may be aqueous or non-aqueous based and employed as foams, gels, suspensions, emulsifiable concentrates, and the like. Ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

As will be appreciated by those skilled in the art, the insecticidal concentration will vary widely with the nature of the particular formulation, particularly whether it is a concentrate or is to be used directly. The pesticide will be present at least 1% by weight, and may be 100% by weight. Dry formulations will have about 1-95% (by weight) insecticide, while liquid formulations will generally be about 1-60% (by weight) solids in the liquid phase. Formulations will generally have from about 10 ² to about 10 ⁴ cells/mg. These formulations will be applied from about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the Lepidoptera pest, eg foliage or soil, by spraying, dusting, sprinkling or the like.

plant transformation . A preferred recombinant host for production of the pesticidal proteins of the invention is a transformed plant. A gene encoding a Bt toxin protein as disclosed herein can be inserted into a plant cell using a variety of techniques well known in the art. For example, a large number of cloning vectors containing the replication system in Escherichia coli and markers allowing selection of transformed cells are available for ready insertion of foreign genes into higher plants. For example, vectors include pBR322, pUC series, M13mp series, pACYC184, and the like. Thus, a DNA fragment having a sequence encoding a Bt toxin protein can be inserted into the vector at an appropriate restriction site. The resulting plasmid can be used to transform E. coli. E. coli cells are grown in a suitable nutrient medium, then harvested and lysed. Plasmids were recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biology methods are generally performed as analytical methods. After each manipulation, the DNA sequence used can be cut and ligated with the next DNA sequence. Each plasmid sequence can be cloned in the same or another plasmid. Other DNA sequences may be necessary depending on the method of inserting the desired gene into the plant. If eg Ti or Ri plasmids are used to transform plant cells, then at least the right border, but often the right and left borders of the T-DNA of the Ti or Ri plasmid must be joined as flanking regions of the gene to be inserted. The use of T-DNA to transform plant cells has been thoroughly studied and well documented in EP 120 516, Lee and Gelvin (2008), Hoekema (1985), Fraley et al., (1986), and An et al., (1985), and is present in well established in the field.

Once the inserted DNA is integrated in the plant genome, it is relatively stable. Transformation vectors typically contain a selectable marker that confers resistance to an antimicrobial or antibiotic such as Bialaphos, kanamycin, G418, bleomycin, or hygromycin, among others, to the transformed plant cell. Thus, individually employed markers should allow selection of transformed cells rather than cells that do not contain the inserted DNA.

A number of techniques are available for inserting DNA into plant host cells. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistic (microparticle bombardment), or electroporation and other possible methods . If Agrobacterium is used for transformation, the DNA to be inserted must be cloned into a special plasmid, either into an intermediate vector or into a binary vector. The intermediate vector can be integrated into the Ti or Ri plasmid by homologous recombination due to sequences homologous to those in the T-DNA. The Ti or Ri plasmid also contains the vir region necessary for the transfer of T-DNA. Intermediate vectors cannot replicate themselves in Agrobacterium. Transfer of the intermediate vector into Agrobacterium tumefaciens (conjugation) can be done by means of a helper plasmid. Binary vectors are self-replicating in both E. coli and Agrobacterium. They contain a selectable marker gene and a linker or polylinker framed by right and left T-DNA border regions. They can be transformed directly into Agrobacterium (Holsters et al., 1978). Agrobacterium used as a host cell contains a plasmid carrying the vir region. The vir region is required for the transfer of T-DNA into plant cells. Can contain other T-DNA. The thus transformed bacteria are used to transform plant cells. Advantageously, plant explants may be cultured with A. tumefaciens or A. rhizogenes to transfer DNA into plant cells. The plant material (e.g., leaf pieces, stalk segments, roots, but also protoplasts or suspension cultured cells) can then be autoinfected in a selection medium that may contain an optional antibiotic or antimicrobial agent Regenerate whole plants. The plants thus obtained can then be tested for the presence of the inserted DNA. Plasmids are not specifically required in the case of injection and electroporation. It is possible to use common plasmids, such as for example pUC derivatives.

Transformed cells grow inside plants in the usual manner. They can form germ cells and pass the transformed traits on to progeny plants. Such plants can be grown in the normal manner and crossed with plants having the same transforming or other genetic factors. The resulting hybrid individuals have corresponding phenotypic properties.

In a preferred embodiment of the invention, plants will be transformed with a gene in which the codon usage has been optimized for the plants. See, eg, US Patent No. 5,380,831, which is hereby incorporated by reference. While some truncated toxins are exemplified herein, it is well known in the Bt art that 130 kDa type (full length) toxins have an N-terminal half as the core toxin and a C-terminal half as the "tail" of the protoxin. Thus, suitable "tails" can be used with the truncated/core toxins of the invention. See, eg, US Patent No. 6,218,188 and US Patent No. 6,673,990. Additionally, methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007). A non-limiting example of a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a CrylFa protein and further comprising a second plant expressible gene encoding a Vip3Ab protein.

Transfer (or introgression) of CrylFa- and Vip3Ab-determining traits to inbred maize lines can be achieved by recurrent selective breeding, for example by backcrossing. - Crossing of donor inbreds (non-recurrent parents) of the genes that determine the trait. Then, the offspring of this cross are backcrossed (mate back) with the recurrent parents, and the desired offspring to be transferred from the non-recurrent parents are then selected among the resulting offspring After 3, preferably 4, more preferably 5 or more generations of backcrossing with the recurrent parent and selection for the desired trait, the offspring will be heterozygous for the locus controlling the transferred trait but will be identical to the recurrent parent in most or almost all other genes (see e.g. Poehlman and Sleper (1995) Breeding Field Crops, 4th ed., 172-175; Fehr (1987) Principles of Cultivar Development, vol. 1 : Theory and Technique, 360-376).

Insect resistance management (IRM) strategies . For example, Roush et al. outline a 2-toxin strategy, also known as "pyramiding" or "stacking," for the management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353, 1777-1786).

On its website, the U.S. Environmental Protection Agency (epa.gov/oppbppd1/biopesticides/pips/bt_corn_refuge_2006.htm) publishes a list of non-transgenic (i.e., non-transgenic) crops that produce a single Bt protein active against target pests. B.t.) Refuge (part of non-Bt crops/corn) following requirements.

"The specific structuring needs of Bt(Cry1Ab or Cry1F) maize products for corn borer protection are as follows:

Structured shelters: 20% non-lepidopteran Bt maize shelters in the Corn Belt;

50% non-lepidopteran Bt refugia in the cotton belt

Blocks

Internal (i.e., within a Bt field)

Outside (i.e., different fields within 1/2 mile (1/4 mile if possible) of Bt fields to maximize random crossing)

Strip

Strips must be at least 4 rows (preferably 6) wide to reduce the effect of larval motility"

Also, the National Corn Growers Association, on their website:

(ncga.com/insect-resistance-management-fact-sheet-bt-corn)

Similar guidance on shelter needs is also provided. For example:

"Corn borer IRM requirements:

- Plant at least 20% of cornfields with shelter hybrids

- In cotton production areas, shelter must be 50%

-Must be planted within 1/2 mile of shelter hybrids

- Refuge can be grown in strips inside Bt fields; refuge strips must be at least 4 rows wide

- Refuges can be treated with conventional insecticides only when the target insect reaches an economic threshold

- Bt based sprayable insecticides cannot be used on refuge corn

-Proper refuge must be planted on every farm with Bt corn"

As described by Roush et al. (eg, pp. 1780 and 1784 right column), the stacking or pyramiding of two different proteins, each effective against a target pest with little or no cross-resistance, may allow the use of smaller refuges. Roush suggests that for successful stacking, refuge sizes of less than 10% refuges can provide comparable resistance management to about 50% refuges for a single (non-pyramided) trait. For currently available pyramided Bt corn products, the US EPA requires significantly less (typically 5%) structured refuges for non-Bt corn than for single-trait products (typically 20%).

There are various ways of providing the IRM effect of refuge, including various geometric planting patterns in the field (as mentioned above) and seed mixtures in bags, as described by Roush et al. (supra) and U.S. Patent No. 6,551,962 for further discussion.

The above percentages, or similar refuge ratios, can be used for thematic double or triple stacks or pyramids. For a triple stack with three modes of action against a single target pest, the aim would be 0 refuge (or eg less than 5% refuge). This applies especially to commercial areas—such as those over 10 acres.

All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety to the extent they do not contradict the express teachings of this specification.

As used herein, the terms "a", "an", and "the/the" mean "at least one" unless expressly indicated or implied.

The following are examples illustrating procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

Example

Example 1: Example overview

The following examples are given showing that Vip3Ab1 is active against wild-type larvae of Fall Armyworm (Fall Armyworm) and against a field-collected strain of Spodoptera frugiperda present in Puerto Rico that is resistant to the Bacillus thuringiensis crystal toxin Cry1Fa . This biological data supports the utility of Vip3Ab1 to be used to combat the development of Cry1 resistance in insects, since insects that develop resistance to the Cry1Fa toxin will continue to be susceptible to the Vip3Ab1 toxin.

Similarly, in Spodoptera frugiperda, ^125I radiolabeled CrylFa binds receptor proteins, and non-radiolabeled CrylFa can be used to displace binding. However, Vip3Ab1 was unable to displace the binding of ¹²⁵ ICry1Fa from its receptor in these experiments. These results indicate that Vip3Ab1 has a unique binding site compared to Cry1Fa. The ability of Vip3Ab1 to exert toxicity against Cry1Fa-resistant insects is based on its demonstrated non-interaction at the site of binding of these toxins. Additional data are presented showing that the Cry1Fa resistance property in Spodoptera frugiperda is due to the inability of Cry1Fa to bind BBMVs prepared from this insect. The biological activity of Vip3Ab1 against Cry1Fa-resistant Spodoptera frugiperda larvae that have lost their ability to bind Cry1Fa further supports a non-interacting target site for Vip3Ab1 compared to Cry1Fa.

Example 2: Purification and trypsin processing of Cry1Fa and Vip3Ab1 proteins.

Genes encoding the Cry1Fa and Vip3Ab1 protoxins were expressed in Pseudomonas fluorescens expression strains and the full-length proteins were isolated as insoluble inclusion bodies. Washed inclusion bodies were dissolved by agitation for 2 hours at 37°C in a buffer containing 20 mM CAPS buffer, pH 11, +10 mM DDT, +0.1% 2-mercaptoethanol. The solution was centrifuged at 27,000 xg for 10 minutes at 37°C, and the supernatant was treated with 0.5% (w/v) TCPK-treated trypsin (Sigma). This solution was incubated with mixing for an additional 1 hour at room temperature, filtered and loaded onto a Pharmacia MonoQ1010 column equilibrated with 20 mM CAPS pH 10.5. After washing the loading column with 2 column volumes of buffer, the truncated toxin was eluted in 15 column volumes using a linear gradient from 0 to 0.5 M NaCl in 20 mM CAPS at a flow rate of 1.0 ml/min. The purified trypsin truncated Cry protein was eluted at about 0.2-0.3M NaCl. The purity of the protein was checked by SDS PAGE and visualized using Coomassie brilliant blue dye. In some cases, the combined fractions of purified toxins were concentrated and loaded onto a Superose 6 column (1.6 cm diameter, 60 cm length) and further purified by size exclusion chromatography. Fractions containing a single peak of monomeric molecular weight were combined and concentrated to yield a preparation that was greater than 95% homogeneous in protein with a molecular weight of approximately 60,000 kDa.

Processing of Vip3Abl was achieved in a similar manner starting with the purified full-length 85 kDa protein (DIG-307) provided by Monte Badger. The protein (12 mg) was dialyzed into 50 mM sodium phosphate buffer, pH 8.4, then processed by adding 1 mg of solid trypsin and incubating for 1 hour at room temperature. The solution was loaded onto a MonoQ anion exchange column (diameter 1 cm, length 10 cm.) and eluted with a linear gradient of 0 to 500 mM NaCl in 20 mM sodium phosphate buffer, pH 8.4, over 7 column volumes. Protein elution was monitored by SDS-PAGE. The major processed band had a molecular weight of 65 kDa as determined by SDS-PAGE using molecular weight standards for comparison.

Example 3: Insect Bioassay.

The purified protein was tested for insecticidal activity in a bioassay with neonatal Spodoptera frugiperda (J.E. Smith) larvae fed an artificial insect diet. Cry1F resistant FAWs were collected from Herculex I (Cry1Fa) corn fields in Puerto Rico and brought to Dow AgroSciences Insectary for further rearing. The characterization of this strain resistant to FAW is outlined in an internal report by Schlenz et al. (Schlenz et al., 2008).

Insect bioassays were performed in 128-well feed bioassay plates (CD International, Pitman, NJ). Each well contained 0.5 mL of multi-species Lepidoptera diet (Southland Products, Lake Village, AR). 40 μL aliquots of purified Cry or Vip3Abl protein diluted at various concentrations in 10 mM CAPS, pH 10.5 or a control solution were delivered by pipetting onto a 1.5 cm diet surface (26.7 μL/cm ^{2 )} per well . 16 wells were tested per sample. The negative control is a buffer solution blank without protein. Positive controls included preparations of Cry1F. The treated dishes were kept in the fume hood until the liquid on the diet surface had evaporated or absorbed into the diet.

Within a few hours of eclosion, individual larvae were picked with a moistened camel hair brush and deposited on the treated diet, 1 larva per well. Infected wells were then sealed with a clear plastic adhesive sheet (C-D International, Pitman, NJ) that was perforated to allow gas exchange. The bioassay dish was maintained under controlled environmental conditions (28 °C, approximately 40% RH, 16:8 [L:D] photoperiod). After 5 days, the total number of insects exposed to each protein sample, the number of dead insects and the weight of surviving insects were recorded.

Example 4: Iodination of CrylFa toxin.

Iodination of Cry1F has been reported to disrupt both toxicity and binding capacity of this protein when tested against tobacco aphid (tobacco budworm) larvae and BBMVs prepared from these insects (Luo et al., 1999; Sheets and Storer, 2001). Inactivation is presumably due to the requirement for an unmodified tyrosine residue near its binding site. Upon iodination of Cry1F using the iodine bead method, the protein lost all its ability to exhibit specific binding characteristics achieved using BBMV from H. virescens. Non-radioactively labeled NaI was used to iodide Cry1F by the iodine bead method, and the iodized Cry1F also lost its insecticidal activity against S. fumigatus.

Earlier studies in our laboratory demonstrated that Cry1Fa could be fluorescently labeled using a maleimide-conjugated labeling reagent that specifically alkylates proteins at cysteine residues. Because the Cry1Fa trypsin core toxin contains a single cysteine residue at position 205, labeling proteins with such reagents results in the alkylation of the protein at a single specific site. It was determined that Cry1Fa could be fluorescently labeled with fluorescein-5-maleimide and that the labeled protein retained insecticidal activity. Based on the retention of the biological activity of cysteine-fluorescein-labeled Cry1Fa, it was determined that we could also radioiodinate the fluorescein moiety of the label by the method of Palmer et al. (Palmer et al., 1997) and attach it to Attached to the cysteine of Cry1Fa, and has radiolabeled Cry1Fa that retains biological activity.

Fluorescein-5-maleimide was dissolved in DMSO at 10 mM (4.27 mg/ml) and then diluted to 1 mM in PBS as determined by its molar extinction coefficient of 68,000 M ⁻¹ cm ⁻¹ . Add 0.5 mCi Na ¹²⁵ I to 70 μl PBS solution containing 2 iodine beads behind a lead shield. The solution was allowed to mix at room temperature for 5 minutes before adding 10 μl of 1 mM fluorescein-5-maleimide. The reaction was allowed to react for 10 minutes, and then the iodine beads were removed. To the reacted solution was added 2 μg of highly purified trypsin-truncated CrylFa core toxin in PBS. Proteins were incubated with iodinated fluorescein-5-maleimide solution for 48 hours at 4°C. The reaction was stopped by adding 2-mercaptoethanol at 14 mM. The reaction mixture was then added to a Zebra spin column equilibrated in 20 mM CAPS, 150 mM KCl, pH 9, and centrifuged at 1,500 xg for 2 minutes to separate unreacted iodinated dye from protein. ^125I radiolabeled fluorescein-Cry1Fa was counted in a gamma counter to determine its specific activity based on an assumed 80% recovery of the input toxin. The protein was also characterized by SDS-PAGE and visualized by phosphorimaging to ensure that the measured radioactivity was covalently associated with the CrylFa protein.

Example 5: Preparation and fractionation of dissolved BBMV.

Standard methods of protein quantification and SDS-polyacrylamide gel electrophoresis were employed, as taught eg in Sambrook et al. (Sambrook and Russell, 2001 ) and updates.

Last instar F. frugiperda larvae were fasted overnight and then dissected after freezing on ice for 15 min. The midgut tissue was removed from the body cavity, leaving the hindgut attached to the body wall. The midgut was dissolved in a 9X volume of ice-cold homogenization buffer (300 mM mannitol, 5 mM GTA, 17 mM Tris base, pH 7.5) supplemented with protease inhibitor cocktail (Sigma-Aldrich P-2714) diluted as recommended by the supplier. )) placed in. Tissue was homogenized with 15 strokes of a glass tissue homogenizer. BBMVs were prepared by the _MgCl2 precipitation method of Wolfersberger (Wolfersberger, 1993). Briefly, an equal volume of 24 mM _MgCl2 solution in 300 mM mannitol was mixed with the midgut homogenate, agitated for 5 min, and allowed to stand on ice for 15 min. The solution was centrifuged at 2,500 xg for 15 minutes at 4°C. The supernatant was retained and the pellet was suspended in the original volume of 0.5X diluted homogenization buffer and centrifuged again. The two supernatants were combined and centrifuged at 27,000 xg for 30 minutes at 4°C to form the BBMV fraction. The pellet was suspended in BBMV storage buffer (10 mM HEPES, 130 mM KCl, 10% glycerol, pH 7.4) at a protein concentration of approximately 3 mg/mL. Protein concentrations were determined using BSA as a standard.

L-leucine-p-nitroaniline aminopeptidase activity (a marker enzyme of the BBMV fraction) was assayed, after which samples were frozen. Briefly, 50 μl of L-leucine-p-nitroaniline (1 mg/ml in PBS) was added to 940 ml of 50 mM Tris HCl in a standard cuvette. The cuvette was placed in a Cary 50 Bio spectrophotometer, zeroed against the absorbance reading at 405 nm, and the reaction was initiated by adding 10 μl of insect midgut homogenate or insect BBMV preparation. The absorbance increase at 405 nm was monitored for 5 minutes at room temperature. The kinetics of the absorbance increase over time during a linear increase in the absorbance of the total protein added to the assay on a per unit basis The specific activities of homogenate and BBMV preparations were determined based on the following equation:

ΔOD/(minute*mg)=aminopeptidase rate (ΔOD/ml*minute)/[protein](mg/ml)

The specific activity of this enzyme is usually 7-fold higher than that present in the starting midgut homogenate fraction. BBMVs were aliquoted into 250 μl samples, snap frozen in _N2 , and stored at –80°C.

Example 6: Electrophoresis

Analysis of proteins by SDS-PAGE was performed under reducing (ie in 5% β-mercaptoethanol, BME) and denaturing (ie heating at 90° for 5 minutes in the presence of 4% SDS) conditions. Proteins were loaded into wells of a 4% to 20% tris-glycine polyacrylamide gel (BioRad; Hercules, CA) and separated at 200 volts for 60 minutes. Protein bands were detected by staining with Coomassie Brilliant Blue R-250 (BioRad) for 1 hr and destained with 5% methanol in 7% acetic acid. Gels were imaged and analyzed using a BioRad Fluro-S Multi Imager ^™ . Relative molecular weights of protein bands were determined by comparison to the mobilities of proteins of known molecular weight observed in a sample of a BenchMark ^™ protein ladder (Invitrogen, Carlsbad, CA) loaded into one well of the gel.

Example 7: Imaging

The radioactive purity of the iodinated Cry1 protein and the measurement of radioactive Cry1Fa in a pull down assay were determined by SDS-PAGE and phosphorimaging. Briefly, after separation and immobilization of proteins by wrapping the gel in Mylar membrane (12 μm thick), the gel was then exposed at least overnight to a Molecular Dynamics storage phosphor screen (35cmx43cm), and SDS-PAGE gels were imaged for up to 4 days. Plates were visualized using a Molecular Dynamics Storm 820 phosphorimager and images were analyzed using ImageQuant ^™ software.

Example 8: Summary of Results

Mortality results from a bioassay of full-length Vip3Abl protein tested at multiple doses against wild-type and CrylFa-resistant Spodoptera frugiperda larvae are shown in Figure 1 . Against wild-type Spodoptera frugiperda larvae, we obtained 100% mortality at the highest concentration tested (9,000 ng/cm ² ), and reduced mortality levels at lower doses. LC-50 was evaluated at approximately 2,000 ng/cm ² . Vip3Ab1 was highly effective against Spodoptera frugiperda in inhibiting larval growth with greater than 95% growth inhibition at concentrations of 1,000 ng/ ^cm2 and higher. The high level of growth inhibition observed for these two species of Spodoptera frugiperda larvae suggested that these insects were most likely to progress to death if maintained for longer periods of time.

A bioassay was also performed to compare the biological activity of Vip3Ab1 against wild type Spodoptera frugiperda versus Cry1Fa resistant Spodoptera frugiperda (Figure 1). Percent growth inhibition is indicated by vertical bars, while percent mortality is indicated by diamond symbols. Mortality measured 5 days after exposure to the toxin was below 50% for both insect types at all concentrations tested. A clear dose response was obtained for growth inhibition. ^Vip3Ab1 resulted in greater than 95% inhibition of larval growth to both Cry1Fa-sensitive and Cry1Fa-resistant L. frugiperda larvae at concentrations above 1,000 ng/cm and to wild ^- type L. frugiperda at about 40 ng/cm. Approximately 50% inhibition of larval growth. Vip3Ab1 resulted in greater than 50% growth inhibition of Cry1Fa resistant Spodoptera frugiperda at all concentrations tested (down to the lowest 4.1 ng/cm ² ). Thus, Vip3Ab1 has high activity against Cry1Fa resistant Spodoptera frugiperda larvae.

Additional bioassay replicates were performed to generate median lethal concentrations (LC50), median growth inhibitory concentrations. Table 2 shows the (GI50) and 95% confidence intervals for Cry1F-susceptible and Cry1F-resistant S. frugiperda for Vip3Ab1 compared to control.

Table 2

A radiolabeled competition binding assay was performed to determine whether Vip3Ab1 interacts at the same site where Cry1Fa binds in FAW. A competition assay was developed to measure the ability of Vip3Ab to compete for the binding ^of125I radiolabeled CrylFa. Figure 2 shows the phosphorimage of radioactive CrylFa separated by SDS-PAGE after binding to BBMV protein. In the absence of any competing ligand, ¹²⁵ ICry1Fa associated with BBMV protein could be detected. Upon incubation in the presence of 1,000 nM unlabeled CrylFa (500-fold more than the concentration of labeled protein used in the assay), very little radioactivity corresponding to ¹²⁵ I CrylFa was detected. Thus, the results show that unlabeled CrylFa competes efficiently with radiolabeled CrylFa for binding to the receptor protein, as would be expected since these cognate proteins bind the same site. When performing the same experiment using 1,000 nM unlabeled Vip3Ab1 protein as a competitor protein, we did not see a change in the level ^of125ICry1Fa binding to the BBMV protein from Spodoptera frugiperda, indicating that Vip3Ab1 did not compete for the binding ^of125ICry1Fa . This result was interpreted as indicating that Vip3Ab1 does not bind at the same site as Cry1Fa.

Insects can develop resistance to Cry protein toxicity via a number of different biochemical mechanisms, but the most common mechanism is due to a reduced ability of Cry toxin proteins to bind to their specific receptors in the insect gut (Heckel et al., 2007; Tabashnik et al., 2007). 2000; Xu et al., 2005). This can be caused via small point mutations, large gene deletions, or via other genetic or biochemical mechanisms. When we investigated BBMV proteins from Cry1Fa-resistant Spodoptera frugiperda to understand the nature of its resistance to Cry1Fa, we found that BBMVs prepared from Cry1Fa-resistant insects were less able to Binding to ¹²⁵ I radiolabeled Cry1Fa (Fig. 3). Thus, the mechanism of resistance to Cry1Fa in Spodoptera frugiperda is due to a greatly reduced level of Cry1Fa binding to BBMV from resistant insects. Since we showed in Figure 2 that Vip3Ab1 does not compete for the binding of Cry1Fa, this further demonstrates that Vip3Ab1 should not be affected by resistance mechanisms involving the binding of Cry1Fa to its specific receptor. This was demonstrated in bioassays. Thus, Vip3Ab1 complements the activity of Cry1Fa because it has biological activity against similar insects, yet does not bind to the same receptor site as these Cry proteins, and as such is not affected by reduced resistance mechanisms that would involve Cry toxin binding. We deduce from these studies that Vip3Ab1 is an excellent insect toxin in combination with Cry1Fa as an insect resistance management method for providing protection against insects that may have developed resistance to either of these proteins. Biologically active, but also against resistant insects.

Reference list

Heckel, D.G., Gahan, L.J., Baxter, S.W., Zhao, J.Z., Shelton, A.M., Gould, F., and Tabashnik, B.E. (2007). The diversity of Bt resistance genes in species of Lepidoptera. J Invertebr Pathol 95, 192-197.

Luo,K.,Banks,D.,and Adang,M.J.(1999).Toxicity,binding,and permeability analyzes of four bacillus thuringiensis cry1 delta-endotoxins using brush border membrane vesicles of spodoptera exigua and spodoptera frugiperda.Appl.Environ.Micro4biol.65, 464.

Palmer, M., Buchkremer, M, Valeva, A, and Bhakdi, S. Cysteine-specific radioiodination of proteins with fluorescein maleimide. Analytical Biochemistry 253, 175-179.1997.

Ref Type: Journal (Full)

Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory).

Schlenz, M.L., Babcock, J.M., and Storer, N.P. Response of Cry1F-resistant and Susceptible European Corn Borer and Fall Armyworm Colonies to Cry1A.105 and Cry12Ab2. DAI 0830, 2008. Indianapolis, Dow AgroSciences. Derbi Report.

Sheets, J.J. and Storer, N.P. Analysis of Cry1Ac Binding to Proteins in Brush Border Membrane Vesicles of Corn Earworm Larvae (Heleothis zea). Interactions with Cry1F Proteins and Its Implication for Resistance in the Field. DAI-0417, 1-26.2001. Indianapolis, Dow Agro .

Tabashnik, B.E., Liu, Y.B., Finson, N., Masson, L., and Heckel, D.G.(1997). Onegene in diamondback moth confers resistance to four Bacillus thuringiensistoxins. Proc. Natl. Acad. Sci. U.S.A 94, 1640- 1644.

Tabashnik, B.E., Malvar, T., Liu, Y.B., Finson, N., Borthakur, D., Shin, B.S., Park, S.H., Masson, L., de Maagd, R.A., and Bosch, D. (1996). Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 62, 2839-2844.

Tabashnik, B.E., Roush, R.T., Earle, E.D., and Shelton, A.M. (2000). Resistance to Bt toxins. Science 287, 42.

Wolfersberger, M.G.(1993).Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgutof the gypsy moth(Lymantria dispar).Arch.Insect Biochem.Physiol 24,139-147.

Xu, X., Yu, L., and Wu, Y. (2005). Disruption of a cadherin gene associated with resistance to Cry1Ac{delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera. Appl Environ Microbiol 71, 948-954.

Appendix A

List of delta-endotoxins – from sites like Crickmore (cited in application)

Accession numbers are NCBI entries

Claims (16)

1. the method generating transgenic plant, it comprises the Vip3Ab insecticidal proteins being made up of by coding SEQ ID NO:2 The DNA of DNA, the DNA of Cry1F insecticidal proteins that coding is made up of SEQ ID NO:1 and coding the 3rd insecticidal proteins be transformed into In host plant, wherein said 3rd insecticidal proteins is selected from Cry1C, Cry1D, Cry1Be and Cry1E.

2. manage the method that autumn mythimna separata forms the resistance to the insecticidal proteins being derived from bacillus thuringiensis, described method bag Include plantation seed to produce multiple plants, the plurality of plant comprises non-Bt refuge plants and multiple transgenic plant, wherein Described transgenic plant comprises the DNA of the Vip3Ab insecticidal proteins that coding is made up of, coding SEQ ID NO:2 by SEQ ID NO: The DNA and the DNA of coding the 3rd insecticidal proteins of the Cry1F insecticidal proteins of 1 composition, wherein said 3rd insecticidal proteins is selected from Cry1C, Cry1D, Cry1Be and Cry1E.

3. the method for claim 2, wherein said multiple plants comprise non-Bt refuge plants and multiple transgenic plant, wherein Described refuge plants account for all crop plants in the plurality of plant less than 40%.

4. the method for claim 3, wherein said refuge plants accounts for being less than of all crop plants in the plurality of plant 30%.

5. the method for claim 3, wherein said refuge plants accounts for being less than of all crop plants in the plurality of plant 20%.

6. the method for claim 3, wherein said refuge plants accounts for being less than of all crop plants in the plurality of plant 10%.

7. the method for claim 3, wherein said refuge plants accounts for being less than of all crop plants in the plurality of plant 5%.

8. the method for claim 3, wherein said refuge plants is (in blocks or strips) in district's group or bar.

9., for controlling the compositions having the autumn mythimna separata lepidoptera pest of resistance to insecticidal proteins, described compositions comprises table Reach the protein containing Cry1F active toxin being made up of SEQ ID NO:1 of insecticidal activity amount, be made up of SEQ ID NO:2 Vip3Ab albumen and the cell of the 3rd insecticidal proteins, described 3rd insecticidal proteins selected from Cry1C, Cry1D, Cry1Be and Cry1E。

10. the compositions of claim 9, its comprise inverted with express by SEQ ID NO:1 form containing Cry1F core poison Protein, the Vip3Ab albumen being made up of SEQ ID NO:2 and the host of the 3rd insecticidal proteins of element, described 3rd parasite killing Albumen is selected from Cry1C, Cry1D, Cry1Be and Cry1E, and wherein said host is microorganism or plant cell.

11. 1 kinds of methods controlling lepidoptera pest, including giving insecticidal activity to described insect or to the environment of described insect The compositions of the claim 9 of amount.

12. 1 kinds manage the method that autumn mythimna separata forms the resistance to Cry toxin, and it is multiple to produce that described method includes planting seed Plant, the plurality of plant comprises non-Bt refuge plants and multiple transgenic plant, and wherein said transgenic plant comprises volume The Cry1F insecticidal proteins that the DNA of the Vip3Ab insecticidal proteins that code is made up of SEQ ID NO:2, coding are made up of SEQ ID NO:1 DNA and the DNA of coding the 3rd insecticidal proteins, wherein said 3rd insecticidal proteins selected from Cry1C, Cry1D, Cry1Be and Cry1E。

The method of 13. claim 12, wherein said non-Bt refuge plants accounts for all crop plants in the plurality of plant Less than 10%.

Method any one of 14. claim 12-13, wherein said multiple plants comprise less than 5% non-Bt refuge plants.

15. the process of claim 1 wherein that described host plant is selected from lower group: corn and soybean and Cotton Gossypii.

16. the process of claim 1 wherein that described plant is corn plant.